As a class of phytohormones, auxins influence virtually every developmental program in plants. Examples of auxins include indole-3-acetic acid (IAA) which is synthesized from tryptophan, indole or indole-3-glycerol phosphate via multiple parallel biochemical pathways (reviewed in Bartel, 1997); indole-3-butyric acid in pea (Schneider et al., 1985) which can be converted to IAA in vivo (Nordstrom et al., 1991); and 4-chloroindole-3-acetic acid (4-Cl-IAA) which has been identified in a number of legumes but seems to be restricted to only certain genera including Pisum sativum (Marumo et al., 1968; Katayama et al., 1988) and Vicia amurensis (Katayama et al., 1987), but not in the closely related Phaseoleae genus (Katayama et al., 1987).
Auxins play vital roles in the coordination of seed and fruit growth in pea. The presence of viable, developing seeds is a prerequisite for pericarp development. Seed removal early in fruit development retards pericarp growth, eventually leading to pericarp senescence (Ozga et al., 1992). Mounting evidence supports the hypothesis that seed-derived signals promote pericarp growth in pea. While the application of bioactive GA3 or GA1 to the endocarp of deseeded pericarps can stimulate pericarp growth (Ozga and Reinecke, 1999), GA transport from the seeds to the pericarp is likely minimal. In the pea GA biosynthesis mutant na, which possesses a loss of function mutation in an ent-kaurene oxidase gene (PsKO1) primarily expressed in vegetative tissues (producing a severely dwarfed plant), the presence of the wildtype seed expressed PsKO2 homolog allows seeds of na mutant plants to develop with normal GA levels, while GA levels in the pod are severely reduced (Davidson et al., 2003). A similar lack of apparent seed to pericarp GA transport was observed in the ls-1 GA biosynthesis mutant (partial loss of the ability to convert GGDP to CPP early in the GA biosynthesis pathway), where pericarp GA1 levels were significantly lower than in wildtype plants while seed GA1 was comparable to wildtype levels (Reid and Ross, 1993).
While transport of bioactive GAs from the seeds to the pericarp as a growth-inducing signal are likely minimal, 4-Cl-IAA can substitute for seeds in many aspects of pericarp growth, and may be a primary seed-to-pericarp growth signal in pea. 4-Cl-IAA accumulates in both the seeds and pericarps of pea (Magnus et al., 1997), and in the absence of viable seeds, 4-Cl-IAA can stimulate pericarp growth (Reinecke et al., 1995). 4-Cl-IAA-stimulated pericarp growth is mediated partially by GAs through the local upregulation of the GA biosynthesis pathway in the pericarp. While [14C]GA12 is efficiently metabolized to GA19 and GA20 by pericarps with intact seeds, in deseeded pericarps [14C]GA19 accumulates, but [14C]GA20 does not, indicating that seeds have a role in the conversion of GA19 to GA20 within the pericarp (Ozga et al., 1992). A similar pattern was observed in the profile of endogenous GAs in the pericarp. In pericarp with seeds, GA19, GA20, GA1, and GA8 were detected; however, in deseeded pericarps, GA19 accumulated, while GA20, GA1, and GA8 were not detected, suggesting a block in the GA pathway at the oxidation step between GA19 and GA20 due to seed removal (Ozga et al., 1992). When applied to the pericarp via a split-pericarp technique, [14C]GA19 was readily converted to [14C]GA20 and [14C]GA29 in fruit with seeds, while in deseeded pericarps, the production of [14C]GA20 and [14C]GA29 was reduced (van Huizen et al., 1995). Steady-state pericarp transcript abundance of PsGA20ox1, the enzyme product of which can convert GA19 to GA20, was lower in deseeded pericarps than in pericarps with seeds, confirming the results of the [14C]GA12 metabolism studies which indicate that seeds are important for pericarp GA20 biosynthesis (van Huizen et al., 1997).
The application of 4-Cl-IAA to deseeded pericarps stimulated the conversion of radiolabelled GA12 or GA19 to GA20 (van Huizen et al., 1995; Ozga et al., 2009), and increased steady-state transcript levels of PsGA20ox1 (van Huizen et al., 1997; Ozga et al., 2009), mimicking the presence of the seeds. In addition to promoting the production of pericarp GA20, seeds are also involved in the regulation of GA 3β-hydroxylase activity, as indicated by the reduction of steady-state PsGA3ox1 in deseeded pericarps in comparison to controls with viable seeds. The application of 4-Cl-IAA to deseeded pericarps once again increased steady-state PsGA3ox1 mRNA levels (Ozga et al., 2003), much as with PsGA20ox1. Additionally, upon treatment with 4-Cl-IAA, deseeded pericarps were able to convert [14C]GA12 to [14C]GA1, which did not occur in the absence of 4-Cl-IAA treatment, indicating the restoration of GA pathway flux in the pericarp by this auxin (Ozga et al., 2009). Transcript abundance of the catabolic gene PsGA2ox1 was elevated in pericarps lacking seeds, and 4-Cl-IAA, but not IAA, reduced PsGA2ox1 transcript to levels comparable to those in pericarps containing viable seeds (Ozga et al., 2009). Between 2 and 3 days after anthesis (DAA), pericarps with viable seeds displayed a transitory increase in transcript abundance of the catabolic gene PsGA2ox2, possibly as part of a regulatory mechanism to support the transition between developmental programs of fruit set and sustained pericarp growth. While PsGA2ox2 transcript levels do not increase in deseeded pericarps, the application of 4-Cl-IAA (but not IAA) to the pericarp can mimic the seed-induced transitory increase in pericarp PsGA2ox2 transcript (Ozga et al., 2009).
The presence of two natural auxins with varying developmental roles provides a unique system in which to study the relationship between physiological activity and auxin structure. Using a split-pericarp pod elongation assay, Reinecke et al. (1995) tested the ability of a variety of halogenated auxins (4-, 5-, 6-, and 7-chloro- and fluoroindole-3-acetic acid) to promote deseeded pericarp growth. While 4-Cl-IAA stimulated pericarp growth, the other auxins tested generally did not stimulate growth. Similar research using a variety of 4-substituted auxins (4-H-IAA, 4-Cl-IAA, 4-Fl-IAA, 4-Me-IAA, and 4-Et-IAA) found that 4-Me-IAA was also capable of stimulating the expansion of deseeded pericarps, but not to the same extent as 4-Cl-IAA (Reinecke et al., 1999). Recent studies of structure-activity relationships in pea pericarp suggests that the position, size, and lipophilicity of the indole-substituent are important for determining biological activity, with optimal activity obtained with a hydrophobic substituent of approximately the same size as a chlorine atom at the 4-position of the indole ring (Reinecke et al., 1999).
Auxins occur in plants as free acids and in conjugated forms. Auxin conjugates include auxin linked to single amino acids or to mono- or disaccharides (Bandurski et al., 1995). IAA can be covalently bound to proteins (Bialek and Cohen, 1989). Auxin conjugation has been implicated as a storage mechanism, where, in addition to de novo synthesis, free auxin can be generated upon cleavage from these bound forms (Bandurski et al., 1995; Woodward and Bartel, 2005). Conjugated auxins in pea include amide conjugates such as indole-3-acetylaspartic acid (Law and Hamilton, 1982) and esterified compounds, such as 1-O-indole-3-acetyl-β-D-glucose (Jakubowska and Kowalczyk, 2004). The ratio between amide and ester conjugates varies between tissues (Bandurski and Schulze, 1977; Magnus et al. 1997), suggesting a developmental role for auxin conjugation in pea. In pea, 4-Cl-IAA has been implicated as a fruit growth promoting auxin (Reinecke et al., 1995; Reinecke et al., 1999; Ozga and Reinecke, 2003).
Most research in auxin signalling has been performed in Arabidopsis. Shortly after application of auxins to Arabidopsis seedlings, a group of transcriptional repressors (the Aux/IAA genes) are upregulated (Leyser, 2002). Aux/IAA proteins are transcriptional repressors and contain an N-terminal transcriptional repressor called domain I (Tiwari et al., 2001), domain II, involved in protein stability and degradation (Park et al., 2002), and two C-terminal dimerization domains III and IV.
Auxin Response Factors (ARF) are similar to the Aux/IAA proteins in structure (Ulmasov et al., 1999), and contain an N-terminal DNA-binding domain, an RNA polymerase II interaction domain (Hagen and Guilfoyle, 2002), and two dimerization domains similar in structure to domains III and IV of the Aux/IAA repressors. The DNA-binding domain recognizes a sequence that consists minimally of a conserved sequence (5′-TGTCTC). This sequence, combined with a secondary constitutive element in some genes (Ulmasov et al., 1995), constitutes the auxin responsive element (ARE), which is necessary and sufficient to confer auxin inducibility to reporter genes. While the Aux/IAA proteins are transcriptional repressors, ARFs can act as transcriptional repressors or activators (Hagen and Guilfoyle, 2002). These two groups of proteins are capable of both homo- and heterodimerization freely with one another. In the absence of auxin, a heterodimer consisting of one Aux/IAA repressor and one ARF protein (either a repressor or an activator) is bound at the ARE of an auxin-inducible gene, inhibiting transcription. Upon auxin induction, the Aux/IAA protein of that dimmer is degraded, which allows the formation of a new homo- or heterodimer, effecting changes in gene transcription.
The degradation of Aux/IAA proteins relies on the SCF complex composed of Skp1, Cullin, and F-box (Gray et al., 1999; FIG. 1). The SCF complex is an E3 ubiquitin ligase involved in several signal transduction pathways, including those for gibberellin and jasmonic acid. Skp1 is a scaffold protein, and interacts with two of the other complex members. Cullin transfers ubiquitin subunits from an E2 ubiquitin conjugating enzyme to a specific target protein, and functions as a heterodimer with a fourth protein, RBX1. The F-box proteins are a diverse family of proteins containing a protein-protein interaction domain which interacts with Skp1 called the F-box, and a variety of C-terminal protein-protein interaction domains which confer target specificity to the complex (leucine rich repeats for the AFB family of F-box proteins (Gagne et al., 2002), although a variety of other domain types are present in other groups of F-box proteins).
In addition to contributing target specificity to the SCF complex, the F-box proteins TIR1, AFB2, and AFB3, function as auxin receptors (Dharmasiri et al., 2005a). The AFB F-box proteins bind auxins directly, and the formation of the auxin-AFB complex is necessary for the binding of Aux/IAA proteins by the SCF (Kepinski and Leyser, 2005). The crystal structure of the TIR1 protein in Arabidopsis in the presence and absence of auxin was obtained (Tan et al., 2007). While the F-box region of the AFB proteins interact with the SCF scaffold protein (ASK1 in Arabidopsis), the C-terminal LRRs form an open pocket. The auxin molecule sits in the proximal end of the pocket and acts as a molecular glue, mediating contact between the AFB protein and the targeted Aux/IAA protein. This binding is likely promoted by van der Waals, hydrophobic, and hydrogen-bonding interactions, and may explain why a number of relatively hydrophobic molecules of approximately the same size and general structure can serve as auxins.
Upon the introduction of auxin into the nucleus, events unfold which culminate in the alteration of transcription profiles of auxin-regulated genes. Initially, auxin binds to the LRR region of the AFB protein of the SCF complex. The auxin molecule mediates interactions between the AFB protein of the SCF complex and the target Aux/IAA protein, which may be part of an inhibitory Aux/IAA-ARF heterodimer. The Cullin subunit of SCF then transfers, iteratively, ubiquitin peptides from E2 ubiquitin conjugating enzymes to a site in domain II of the Aux/IAA protein (Dharmasiri and Estelle, 2004). The ubiquitinated Aux/IAA protein is shuttled to the 26s proteasome for degradation (Gray et al., 2001), freeing the formerly bound ARF protein to interact with other subunits. Another ARF subunit or a second Aux/IAA protein (if more are available) can then dimerize with the pre-existing ARF protein, either promoting or inhibiting transcription of the auxin-responsive gene, leading to a variety of physiological and developmental changes (Dharmasiri et al., 2005b; FIG. 2). In the absence of an appropriate auxin, an ARF-Aux/IAA heterodimer binds the upstream ARE sequence, preventing transcription. Upon degradation of the Aux/IAA protein by the auxin-activated SCF complex, an ARF homodimer can form, recruiting RNApol II and increasing transcription.